本文選題：H9N2 + AIV　； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：H9N2亞型禽流感為低致病性禽流感(Low Pathogenic Avian influenza,LPAI),其傳染性較高、致死率較低,感染后可造成禽類的免疫力下降,呼吸道和生殖道系統(tǒng)的病變,蛋雞表現(xiàn)為產(chǎn)蛋率顯著下降,蛋的品質(zhì)下降,產(chǎn)薄殼蛋、軟殼蛋、砂皮蛋和畸形蛋,引發(fā)嚴(yán)重的經(jīng)濟(jì)損失。但目前尚未見關(guān)于H9N2亞型禽流感病毒感染如何導(dǎo)致雞輸卵管上皮細(xì)胞發(fā)生病變的分子機制研究的報道。血紅素加氧酶1(Heme Oxygenase-1,HO-1)為機體內(nèi)重要的抗氧化應(yīng)激蛋白酶,在抗炎、抗氧化、抗細(xì)胞凋亡、抗病毒感染中都有重要的作用。因此,本實驗分離培養(yǎng)了原代雞輸卵管上皮細(xì)胞(chicken oviduct epithelial cells,COEC),研究了H9N2 AIV感染對細(xì)胞中HO-1表達(dá)的影響,并初步探索HO-1調(diào)控H9N2 AIV增殖的機制,主要研究結(jié)果如下:1.H9N2 AIV在雞輸卵管上皮細(xì)胞中的增殖研究。成功分離培養(yǎng)了雞輸卵管上皮細(xì)胞,細(xì)胞表現(xiàn)為典型的上皮樣形態(tài),生長狀態(tài)良好,角蛋白18鑒定呈陽性。H9N2 AIV感染第3代COEC,可觀察到細(xì)胞產(chǎn)生明顯的CPE,表現(xiàn)為細(xì)胞皺縮、拉長變細(xì),部分細(xì)胞變圓,甚至裂解死亡。Western Blot可檢測到宿主細(xì)胞中有病毒NS1蛋白的表達(dá)。2.H9N2 AIV感染COEC后細(xì)胞內(nèi)HO-1的表達(dá)變化。H9N2 AIV感染COEC后,通過Real time PCR及Western Blot方法檢測不同時間點HO-1的變化情況。結(jié)果表明,HO-1的mRNA和蛋白表達(dá)均水平顯著增加。3.HO-1在H9N2 AIV的增殖過程中的作用研究。為了研究HO-1在H9N2 AIV增殖過程中的作用,我們首先用HO-1的抑制劑ZnPP處理COEC,Western Blot檢測ZnPP抑制HO-1對H9N2 AIV增殖的影響。結(jié)果表明,ZnPP抑制了細(xì)胞中HO-1表達(dá)的同時,也顯著抑制H9N2 AIV的增殖水平。之后,我們將能特異性干擾HO-1表達(dá)的siRNA轉(zhuǎn)染COEC,同樣顯著降低了HO-1和H9N2 AIV的蛋白表達(dá)水平。綜上所述,本研究表明H9N2 AIV感染可上調(diào)COEC中HO-1的表達(dá),而下調(diào)HO-1表達(dá)可顯著抑制H9N2 AIV在細(xì)胞中的增殖。研究結(jié)果為抗H9N2藥物的研發(fā)提供重要的參考靶點,也為禽流感病毒的防治提供了新思路。
[Abstract]:H9N2 subtype avian influenza (H9N2) is a low pathogenic Avian influenzavirus (LPAI) with high infectivity and low mortality. It can cause the decrease of poultry immunity and the pathological changes of respiratory tract and reproductive system after infection. The laying rate of laying hens is significantly decreased. The quality of eggs declined, and the production of thin shell eggs, soft shell eggs, sand eggs and malformed eggs caused serious economic losses. However, there are no reports on the molecular mechanism of H9N2 subtype avian influenza virus infection in the pathogenesis of oviduct epithelial cell lesions in chickens. Heme Oxygenase-1 (Heme Oxygenase-1) is an important antioxidant stress protease, which plays an important role in anti-inflammation, anti-oxidation, anti-apoptosis and anti-virus infection. Therefore, we isolated and cultured primary chicken oviductal epithelial cells (chicken oviduct epithelial cells), studied the effect of H9N2 AIV infection on the expression of HO-1, and preliminarily explored the mechanism of HO-1 regulating H9N2 AIV proliferation. The main results are as follows: 1. The proliferation of H 9 N 2 AIV in chicken oviduct epithelial cells. Chicken oviduct epithelial cells were isolated and cultured successfully. The cells showed typical epithelial-like morphology and good growth state. The positive reaction of keratin 18 was observed in the third generation of COEC.H9N2 AIV infection. The expression of virus NS1 protein in host cells was detected by Western Blot. 2. The changes of HO-1 expression in host cells after infection with COEC. H9N2 AIV infected COEC. Real time PCR and Western blot were used to detect the changes of HO-1 at different time points. The results showed that the mRNA and protein expression of HO-1 increased significantly. 3. The role of HO-1 in the proliferation of H9N2 AIV was studied. In order to study the role of HO-1 in the proliferation of H9N2 AIV, we first detected the effect of inhibition of HO-1 on H9N2 AIV proliferation by ZnPP treatment with COEC-Western Blot. The results showed that ZnPP inhibited the expression of HO-1 and significantly inhibited the proliferation of H9N2 AIV. Subsequently, we transfected COECs with siRNA that specifically interfered with HO-1 expression, which also significantly reduced the protein expression level of HO-1 and H9N2 AIV. In conclusion, H9N2 AIV infection can up-regulate the expression of HO-1 in COEC, but down-regulate HO-1 expression can significantly inhibit the proliferation of H9N2 AIV in cells. The results provide an important reference target for the development of anti-H9N2 drugs and a new idea for the prevention and treatment of avian influenza virus.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.31

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